Attention recently has been focused on the biological activities of the proteolytically-processed polypeptides from post-translational modified peptide hormones. For example, the C-peptide of pro-insulin has long been regarded to be biologically inactive except for a possible role in the folding of the insulin molecule during its post-translational modification. However, Ito et al. (14) have found recently that the C-peptide of pro-insulin is important in restoring vascular and neural dysfunction and Na+/K+-dependent ATPase activity in diabetic rats. Although a synthetic peptide amide of human b-type IGF-I E-peptide has been shown to exert mitogenic activity by Siefried et al. (5), the biological activity of the native human E-peptides have not been identified.
Insulin-like growth factor-I (IGF-1) is a member of the insulin gene family and plays an essential role in growth, differentiation, regeneration and metabolism in all vertebrates (2). In all animals studied to-date, the primary translation product of IGF-I mRNA contains distinct regions or domains, including an N-terminal pre-peptide leader, followed by the mature peptide with B, C, A and D domains, and a C-terminus E domain. Due to alternative RNA splicing, two different forms, the a- and b-type, of IGF-I pro-hormones exist in mammals, differing only in the E-peptide region. In human, mouse and rat, the first 15 amino acid residues in the N-termini of the E-peptides of pro-IGF-Ia and -Ib share identical sequences, and this region is referred to as the common region. The sub-types of pro-IGF-I's are distinguished from one another by the amino acid sequences after the first 15 common amino acids, known as the variable regions of the E-peptides (3).
In mammals, the signal peptide and the E-peptide are proteolytically cleaved after translation from the pre-pro-hormone to form a mature peptide of 70 amino acid residues. The E-peptides of mammalian pro-insulin-like growth factor-I (pro-IGF-I) have long been regarded as biologically inactive.
Surprisingly, the present inventors have recently discovered that certain recombinant rainbow trout pro-IGF-I E-peptides (Ea-2-, Ea-3- and Ea-4), like hIGF-I, exhibit a dose-related mitogenic activity in several mammalian cell lines (Tian et al. (1)).
Four different forms of a-type IGF-I mRNA have been observed in rainbow trout (rt), Oncorhynchus mykiss, and are designated as IGF-I Ea-1, Ea-2, Ea-3 and Ea-4 (4). Nucleotide sequence comparison of the four size forms of IGF-I mRNAs reveal that the size differences among these mRNA species are due to insertions or deletions in the E domain regions of the molecules. The first 15 predicted amino acid residues of the four E-peptides share identical sequences among themselves as well as with pro-IGF-I E-peptides of human, mouse, and rat. The presence of the C-terminal 20 amino acid residues, sharing 70% identity with their human counterparts, identifies them to the a-type E-peptides.
The Ea-1-peptide of the rt-pro-IGF-I is a polypeptide of 35 amino acid residues, comprising the first 15 and the last 20 amino acid residues. Ea-2 and Ea-3 peptides differ from Ea-1 by virtue of either a 12- or 27-amino acid residue insertion between the first and last segments of the Ea-1-peptide sequence, respectively, while Ea-4 contains both insertions. The predicted numbers of amino acid residues in each E-peptide are, thus, 35, 47, 62 and 74, respectively. There has not been any report on the presence of b-type IGF-I mRNA in rainbow trout.
While the biological functions of the mature IGF-I peptide have been intensively studied, the functions of E-peptides have been overlooked and remain to be identified.
Siegfried et al. (5) have recently shown that the amide of a 22-amino acid residue encoded within the variable region of the E-peptide of the human IGF-Ib (IBE1) exerts mitogenic activity in normal and malignant bronchial epithelial cells. Surprisingly, as set forth above, the present inventors have recently found that certain E-peptides from fish possess mitogenic activity with respect to mammalian cells (Tian et al. (1) report that recombinant rainbow trout Ea-2-, Ea-3- and Ea-4-peptide possess mitogenic activity in several normal and oncogenic transformed mammalian cell lines including NIH 3T3 cells, human embryonic kidney cells transformed by retrovirus (293GP), human mammary gland tumor cells (MCF-7) and caprine mammary epithelium cells (CMEC) (6)).
Based on the surprising inter-class activity of iethyofauna Pro-IGF-I E-peptides on mammalian cells, and given that trout Ea-2, Ea-3 and Ea-4 peptides containing a signal motif for peptidyl C-terminal amidation (4,7) and a bipartite consensus nuclear localization sequence (4,8), the present inventors hypothesized that these peptides might possess other novel biological activities on mammalian cells. In its screening for novel biological activities of trout pro-IGF-1 E-peptides, the present inventors undertook several studies of the effects of such E-peptides on morphology, colony formation activity on a soft agar medium, and invasiveness of human and trout cancer cells. Surprising and unexpected anti-tumor activity was discovered.